The Replication Activity of Influenza Virus Polymerase Is Linked to
the Capacity of the PA Subunit To Induce Proteolysis
"The PA subunit of the influenza virus polymerase complex is a
phosphorylated protein that induces a proteolytic process that
decreases its own accumulation levels and those of coexpressed
proteins. The amino-terminal third of the protein is responsible for
the induction of proteolysis. We mutated five potential casein kinase
II phosphorylation sites located in the amino-terminal third of the
protein. Mutations affecting position 157 almost completely abrogated
proteolysis induction, whereas a mutation at position 162 produced a
moderate decrease and mutations at positions 151, 200, and 224 did not
affect proteolysis induction. Reconstitution of the influenza virus
polymerase in vivo with viral model RNA containing the chloramphenicol
acetyltransferase (CAT) gene indicated that the CAT activity obtained
correlated with the capacity of each PA mutant to induce proteolysis.
RNA protection assays of the products obtained with viral polymerase,
reconstituted in vivo with model RNAs, indicated that mutations at
position 157 led to a selective loss of the ability to synthesize cRNA
from the viral RNA template but not to transcribe viral RNA, while a
mutation affecting position 162 showed an intermediate phenotype.
Collectively, these data provide a link between PA-mediated induction
of proteolysis and the replication activity of the polymerase."
So anyway, I did some searching-
(the dot fna file in the parent directory is 120 MB)
search for influenza on this page:
and in particular get to these 8 sequenced segments of influenzavirus A:
and in particular, segment 3 is polymerase PA:
while segment 2 is polymerase PB1:
while segment 1 is polymerase PB2:
Some issues come to mind. First, viruses and any other
self-replicating system is going to generally be subject to natural
selection, so if you make a blocker for the PA subunit, those
influenza viruses that mutate and are able to get around that PA
subunit inhibitor will be able to obviously get past your antiviral,
and using the antiviral would increase the selective pressure on this.
Now, I suppose I can imagine a scenario where you are able to
simultaneously get the rates down for the flu so low in that these
selective pressures might not "have as much time to kick into action"
(somehow overcoming the natural rate of mutation of influenza, and
whatever benefit that natural rate of mutation may or may not have).
The PB1 subunit alone can catalyze cRNA synthesis, and the PA subunit
in addition to the PB1 subunit is required for viral RNA synthesis in
replication of the influenza virus genome.J. Virol. 1996 Sep;
Crystal structure of the polymerase PAC–PB1N complex from an avian
influenza H5N1 virus
Figure 2c shows locations of known mutations to PAc. "The recent
emergence of highly pathogenic avian influenza A virus strains with
subtype H5N1 pose a global threat to human health1. Elucidation of the
underlying mechanisms of viral replication is critical for development
of anti-influenza virus drugs2. The influenza RNA-dependent RNA
polymerase (RdRp) heterotrimer has crucial roles in viral RNA
replication and transcription. It contains three proteins: PA, PB1 and
PB2. PB1 harbours polymerase and endonuclease activities and PB2 is
responsible for cap binding3, 4; PA is implicated in RNA replication5,
6, 7, 8, 9, 10 and proteolytic activity11, 12, 13, 14, although its
function is less clearly defined. Here we report the 2.9 ångström
structure of avian H5N1 influenza A virus PA (PAC, residues 257–716)
in complex with the PA-binding region of PB1 (PB1N, residues 1–25).
PAC has a fold resembling a dragon's head with PB1N clamped into its
open 'jaws'. PB1N is a known inhibitor that blocks assembly of the
polymerase heterotrimer and abolishes viral replication. Our structure
provides details for the binding of PB1N to PAC at the atomic level,
demonstrating a potential target for novel anti-influenza
therapeutics. We also discuss a potential nucleotide binding site and
the roles of some known residues involved in polymerase activity.
Furthermore, to explore the role of PA in viral replication and
transcription, we propose a model for the influenza RdRp heterotrimer
by comparing PAC with the 3 reovirus polymerase structure, and docking
the PAC structure into an available low resolution electron microscopy
> If you have the flu yourself, you can extract its RNA. Flu is easy to
> grow in eggs, that's how the vaccine is made. (No, you're not able to
Can you describe this some more please, or provide some references?
What type of eggs?
> grow enough flu to start your own personal epidemic in your garage,
> try as you might--but you can get enough RNA to experiment with.) Flu
> has a segmented genome, so getting the individual pieces of nucleic
> acid for each individual protein is as simple as running a gel.
I wonder if just ingesting PB1N ("residues 1-25") would be enough
here- or at least somehow getting into the body and on the scene. In
which case, that would be:
> You can use any expression method of choice to grow the individual
> proteins, and do shifts or Far Westerns to find binding partners. e.g.
> to confirm that it is the PA subunit that steals RNA caps, you could
> incubate the various units of the flu protein with RNA extracted from
> grocery store chicken, and look for proteins that shift upwards in
> size on a gel. Then blot the gel onto nitrocellulose, block with 5%
> milk and probe with a labeled version of the original flu RNA
Right. Sounds like the above study on the replication activity of the
subunits would be a good starting point on that type of home
> Pull-downs to find binding partners or potential inhibitors are easy
> things that can be done at home. If someone could take the straw
> method of electrophoresis and high throughput-ize it, they'd have
> their very own drug discovery lab.
So, the experimental design would be to do some large scale automation
or high throughput work to figure out which mutations cause the virus
replication activity to drop like a rock, from looking at the gel
results. What about actually getting the PAc subunit inhibitor though?
I suppose that one terrible way to do it would be to separate the
genome by mass via gel electrophoresis, cut out the PB1N proteins, and
make a giant library and keep detailed records of where each one came
from for each experiment, trying to find a natural mutation from your
many rounds of replicating the virus' proteins. But maybe there's a
more direct method that might involve, say, ordering some oligos over
> If you have the flu yourself, you can extract its RNA. Flu is easy to
> grow in eggs, that's how the vaccine is made.
Uh, can't the flu virus cause disease in healthy humans? Doesn't that
mean it's at least BSL 2?
Education time. If you're already sick with the flu, how is
cultivating it going to hurt you more?
There's a completely unrelated flu epidemic that kills about 50 million people like the one in 1918, then someone reads this mailing list looking for someone to blame, and sees that you once said "If you're already sick with the flu, how is cultivating it going to hurt you more?"
And then they all come round to your house with flaming torches and pitchforks.
Or there isn't, but for some reason there's a massive panic because someone's created the rumour that this year's flu was actually created in a DIYbio lab by mistake... so loads of people come round to your house with flaming torches and pitchforks.
Or you could actually create a variant that kills 50 million people. Or just one person.
And so on. Use your imagination, because sooner or later someone wanting to sell newspapers is going to use theirs.
That sounds like it would be a fantastically fun kit to build,
distribute and use. The instructions are fairly simple- even PCR could
be made to have a simple protocol and instructions- and then the
antibiotics and resistance tests might have to be purchased from the
pharmacy or drug store down the street (that sort of thing), but other
than that, a package of cotton swabs, some plastic plates with tops,
and some instructions might make for an interesting project. And if
I'm not mistaken, this is what the bioweathermaps project is about-
except getting some deals for individuals to send in DNA samples for
sequencing and subsequent identification (which, by the way, should
probably be confirmed visually by looking at the cultures and culture
Btw, I see two books for Bergey: Bergey's Manual of Systematic
Bacteriology, and Bergey's Manual of Determinitive Bacteriology; which
one are you referring to, or is there a book specifically on micro
that I'm not seeing?
Just so, and well said. Don't confuse two issues:
* Being safe
* Giving others confidence of your work being safe
These are definitely not the same, and you can confuse
yourself (in either direction) if you mix them up.
You need to both be safe and provide others assurance
that you are being safe. Doing BL-2 work at home, while
perhaps safe for some people, does not give the appearance
of being safe. You (and our community!) will suffer.